Does prior exposure to larvicides influence dengue virus susceptibility in Aedes aegypti (Diptera: Culicidae)?

Control of mosquito vector populations is primarily intended to reduce the transmission of pathogens they transmit. Use of chemical controls, such as larvicides, can have unforeseen consequences on adult traits if not applied properly. The consequences of under application of larvicides are little studied, specifically the impacts on pathogen infection and transmission by the vectors that survive exposure to larvicides. We compared vector susceptibility of Aedes aegypti (L.) for dengue virus, serotype 1 (DENV-1) previously exposed as larvae to an LC50 of different classes of insecticides as formulated larvicides. Larval exposure to insect growth regulators (methoprene and pyriproxyfen) significantly increased susceptibility to infection of DENV-1 in Ae. aegypti adults but did not alter disseminated infection or transmission. Larval exposure to temephos, spinosad, and Bti did not increase infection, disseminated infection, or transmission of DENV-1. Our findings describe a previously under observed phenomenon, the latent effects of select larvicides on mosquito vector susceptibility for arboviruses. These data suggest that there are unintended consequences of sublethal exposure to select larvicides that can influence susceptibility of Ae. aegypti to DENV infection, and indicates the need for further investigation of sublethal effects of insecticides on other aspects of mosquito biology, especially those parameters relevant to a mosquitoes ability to transmit arboviruses (life span, biting behavior, extrinsic incubation period).

A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect.

BACKGROUND: Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS: A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS: Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.

Gene editing of the ABC Transporter/White locus using CRISPR/Cas9-mediated mutagenesis in the Indian Meal Moth.

ATP binding cassette (ABC) proteins are involved in transport of substrates across membranes including eye pigments. Mutations of ABC transporter white, brown and scarlet genes of Drosophila and other insects result in visible eye color phenotypes. White locus was identified in a genome assembly of Plodia interpunctella and was found to extend for 16,670 bp comprising 13 exons. We report here recovery of heritable mutants in white in the Indian meal moth, P. interpunctella, using CRISPR/Cas9-mediated mutagenesis. A white eye strain of P. interpunctella c.737delC (Piw-/-) was previously isolated in 1986. Guide RNA (sgRNA) was designed for exon 1 (sgRNA242). Microinjection of Cas9/sgRNA242 complex into Plodia wild type eggs (≤20 min post oviposition) produced 156 viable larvae of which 81 eclosed as adults. Forty-five (56 %) adults displayed wild type phenotype, while 26 females (32 %) and 10 males (12 %) showed full or partial white eye phenotype. The 26 white eye females were mated with Piw-/- males and 21 matings resulted in F1 white eye progeny. Thirteen of the Piw-242 lines were established and sequencing showed indels at the CRISPR/Cas9 242AM site. Based on RT-PCR analysis, most white mutations resulted in suppressed levels of transcript. These results demonstrate the utility of CRISPR/Cas9 gene editing in Plodia which suggests this technology can be used to characterize the role of various genetic elements including those that encode novel targets or confer insecticide resistance mechanisms.

Lethal and Sublethal Concentrations of Formulated Larvicides Against Susceptible Aedes aegypti.

Chemical control of vectors depends on the effective application of formulated insecticides. In this study we evaluated formulated larvicides using a larval bioassay against susceptible Aedes aegypti. The estimated larvicide lethal concentrations for 50% mortality (LC50s) were 25.7 µg/liter (Natular 2EC), 3.13 µg/liter (Abate 4E), 0.43 µg/liter (Altosid), 0.03 µg/liter (Nyguard), and 500.6 ITU/liter (VectoBac12AS containing Bacillus thuringiensis israelensis). Sublethal effects were identified and documented from adults that survived exposure to these estimated LC50s (body size and sex proportion). We observed changes in net growth as measured by adult wing lengths. For those larvae exposed to estimated LC50s, the average size of adults was between 0.1% and 10.6% smaller for males and between 1.1% and 13.6% smaller for females compared to controls. Sex proportions varied between larvicides, but some were significantly different from the control, favoring greater survival of females than males.

RNAi-induced knockdown of white gene in the southern green stink bug (Nezara viridula L.).

The southern green stink bug (SGSB) Nezara viridula L. is one of the most common stink bug species in the United States and can cause significant yield loss in a variety of crops. A suitable marker for the assessment of gene-editing tools in SGSB has yet to be characterized. The white gene, first documented in Drosophila, has been a useful target to assess the efficiency of introduced mutations in many species as it controls pigmentation processes and mutants display readily identifiable phenotypes. In this study we used the RNAi technique to investigate functions and phenotypes associated with the white ortholog in the SGSB and to validate white as a marker for genetic transformation in this species. This study revealed that white may be a suitable marker for germline transformation in the SGSB as white transcript knockdown was not lethal, did not impair embryo development and provided a distinguishable phenotype. Our results demonstrated that the white ortholog in SGSB is involved in the pathway for ommochrome synthesis and suggested additional functions of this gene such as in the integument composition, management of hemolymph compounds and riboflavin mobilization.

Up-regulation of apoptotic- and cell survival-related gene pathways following exposures of western corn rootworm to B. thuringiensis crystalline pesticidal proteins in transgenic maize roots.

BACKGROUND: Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera, being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood. RESULTS: Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae. Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein. CONCLUSIONS: Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.

Influence of Pyrethroid Resistance on Vector Competency for Zika Virus by Aedes aegypti (Diptera: Culicidae).

The vector competence of mosquitoes for pathogens has been shown to be influenced by the status of insecticide resistance in the mosquito population. However, to date, only two studies has explored the impact of insecticide resistance on arbovirus transmission. The global and widespread use of pyrethroids has led to the development of insecticide resistance in many mosquito species, including Aedes aegypti (Linnaeus) (Diptera: Culicidae), the primary vector of Zika virus. Strains of Ae. aegypti that were genetically similar, but responded differently to pyrethroid exposure, were developed using backcrossing techniques. These populations were orally infected with Zika virus and susceptibility to infection, disseminated infection, and transmission potential were evaluated. Analyses revealed differences in susceptibility to infection and disseminated infection between the pyrethroid susceptible and resistant strains of Ae. aegypti during the infection period. Here, we identify an additional challenge to that of widespread pyrethroid resistance. Specifically, resistance is associated with altered phenotypic traits that influence susceptibility to arbovirus infection and progression of infection in the mosquito, factors which ultimately influence risk of arbovirus transmission. These findings support the need to 1) consider insecticide resistance status during times of arbovirus transmission and 2) to implement insecticide resistance management/ mitigation strategies in vector control programs.

The Use of Insecticides to Manage the Western Corn Rootworm, Diabrotica virgifera virgifera, LeConte: History, Field-Evolved Resistance, and Associated Mechanisms.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Dvv) is a significant insect pest of maize in the United States (U.S.). This paper reviews the history of insecticide use in Dvv management programs, Dvv adaptation to insecticides, i.e., field-evolved resistance and associated mechanisms of resistance, plus the current role of insecticides in the transgenic era. In the western U.S. Corn Belt where continuous maize is commonly grown in large irrigated monocultures, broadcast-applied soil or foliar insecticides have been extensively used over time to manage annual densities of Dvv and other secondary insect pests. This has contributed to the sequential occurrence of Dvv resistance evolution to cyclodiene, organophosphate, carbamate, and pyrethroid insecticides since the 1950s. Mechanisms of resistance are complex, but both oxidative and hydrolytic metabolism contribute to organophosphate, carbamate, and pyrethroid resistance facilitating cross-resistance between insecticide classes. History shows that Dvv insecticide resistance can evolve quickly and may persist in field populations even in the absence of selection. This suggests minimal fitness costs associated with Dvv resistance. In the transgenic era, insecticides function primarily as complementary tools with other Dvv management tactics to manage annual Dvv densities/crop injury and resistance over time.

Molecular characterization of western corn rootworm pyrethroid resistance.

BACKGROUND: Western corn rootworm (WCR) pyrethroid resistance has been confirmed in the western US Corn Belt. Toxicological and biochemical studies indicated that multiple mechanisms of resistance might be involved in the resistance trait, such as enhanced metabolism and/or kdr target-site mutation(s) in the voltage-gated sodium channels. To characterize the mechanisms of WCR pyrethroid resistance at the molecular level, pairwise comparisons were made between RNA-Seq data collected from pyrethroid-resistant and -susceptible WCR populations. Gene expression levels and sodium channel sequences were evaluated. RESULTS: Seven transcripts exhibited significantly different expression (q ≤ 0.05) when comparing field-collected pyrethroid-resistant (R-Field) and -susceptible (S-Field) WCR populations. Three of the differentially expressed transcripts were P450s overexpressed in R-Field (9.2-26.2-fold). A higher number (99) of differentially expressed transcripts was found when comparing laboratory-derived pyrethroid-resistant (R-Lab) and -susceptible (S-Lab) WCR populations. Eight of the significant transcripts were P450s overexpressed in R-Lab (2.7-39.8-fold). This study did not detect kdr mutations in pyrethroid-resistant WCR populations. Other differentially expressed transcripts that may play a role in WCR pyrethroid resistance are discussed. CONCLUSION: This study revealed that P450-mediated metabolism is likely to be a major mechanism of WCR pyrethroid resistance, which could affect the efficacy of other insecticides sharing similar metabolic pathways. Additionally, results suggested that although laboratory selection of a pyrethroid-resistant WCR population may help to characterize resistance mechanisms, a field-selected population provided rare and perhaps major variants corresponding to the resistance trait.

Demographic Performance of Helicoverpa zea Populations on Dual and Triple-Gene Bt Cotton.

Insecticidal toxins from Bacillus thuringiensis (Bt) are valuable tools for pest management worldwide, contributing to the management of human disease insect vectors and phytophagous insect pests of agriculture and forestry. Here, we report the effects of dual and triple Bt toxins expressed in transgenic cotton cultivars on the fitness and demographic performance of Helicoverpa zea (Boddie)-a noctuid pest, known as cotton bollworm and corn earworm. Life-history traits were determined for individuals of three field populations from a region where H. zea overwintering is likely. Triple-gene Bt cotton cultivars that express Cry and Vip3Aa toxins killed 100% of the larvae in all populations tested. In contrast, dual-gene Bt cotton that express Cry1Ac+Cry1F and Cry1Ac+Cry2Ab allowed population growth with the intrinsic rate of population growth (rm) 38% lower than on non-Bt cotton. The insects feeding on Bt cotton plants that express Cry1Ac+Cry2Ab, Cry1Ac+Cry1F, or Cry1Ab+Cry2Ae exhibited reduced larval weight, survival rate, and increased development time. Additionally, fitness parameters varied significantly among the insect populations, even on non-Bt cotton plants, likely because of their different genetic background and/or previous Bt toxin exposure. This is the first report of the comparative fitness of H. zea field populations on dual-gene Bt cotton after the recent reports of field resistance to certain Bt toxins. These results document the population growth rates of H. zea from an agricultural landscape with 100% Bt cotton cultivars. Our results will contribute to the development and validation of resistance management recommendations.

Contrasting susceptibility of lepidopteran pests to diamide and pyrethroid insecticides in a region of overwintering and migratory intersection.

BACKGROUND: Pesticide resistance is a growing issue worldwide, and susceptibility of pest populations should be monitored in migratory intersection regions for successful resistance management. We determined the susceptibility of eight noctuid species from the Florida Panhandle to bifenthrin (pyrethroid) and chlorantraniliprole (diamide). Larvae from field and laboratory populations were exposed to commercial insecticide formulations using the leaf-dip method in concentration-mortality bioassays. RESULTS: The field populations of Helicoverpa zea (Boddie), Spodoptera frugiperda (Smith), S. eridania (Stoll), S. exigua (Hubner) and Chloridea virescens (Fabricius) had reduced susceptibility to bifenthrin compared with the laboratory populations. Resistance ratios to bifenthrin were as high as 10 071-fold in S. exigua and 436-fold in S. frugiperda, while there was no reduced susceptibility in Agrotis ipsilon (Hufnagel). The susceptibility to chlorantraniliprole was similar between the field and laboratory populations studied, except for S. exigua that exhibited 630-fold resistance to the diamide. The probit regression equations indicated that the larval mortality of S. exigua and S. frugiperda populations was <80% with bifenthrin at the concentration equivalent to the label rate. Likewise, the estimated mortality of S. exigua larvae with chlorantraniliprole at the label rate concentration was <80%. CONCLUSIONS: The lepidopteran pest populations tested were variable in susceptibility to bifenthrin by contrast to more consistent susceptibility to chlorantraniliprole. These results help in the choice of effective insecticides for integrated pest management and resistance management in cropping systems colonized by migratory lepidopteran pests from the U.S. Gulf Coast region. © 2020 Society of Chemical Industry.

Dietary RNAi toxicity assay exhibits differential responses to ingested dsRNAs among lady beetles.

BACKGROUND: Most recently, major federal regulatory agencies deregulated an in planta RNA interference (RNAi) trait against a devastating corn pest, the western corn rootworm Diabrotica virgifera virgifera, in the United States and Canada. The impact of double-stranded RNA (dsRNA) plant-incorporated protectants (PIPs) and dietary RNAi to non-target organisms, however, still needs further investigation. In this study, we assessed the potential risks of a Diabrotica virgifera virgifera active dsRNA to a group of predatory biological control agents, including Hippodamia convergens, Harmonia axyridis, Coleomegilla maculata, and Coccinella septempunctata. The overarching hypothesis is that the insecticidal dsRNA targeting Diabrotica virgifera virgifera has no or negligible adverse effect on lady beetles. RESULTS: A 400-bp fragment with the highest sequence similarity between target and tested species was selected as the template for dsRNA synthesis. For the dietary RNAi toxicity assay, newly hatched first instar larvae were administered with v-ATPase A dsRNAs designed from Diabrotica virgifera virgifera and the four lady beetles, respectively. A dsRNA from ß-glucuronidase (GUS), a plant gene, and H2 O were served as the negative controls. The endpoint included both sub-organismal (gene expression), and organismal (survival rate, development time, pupa and adult weight) measurements. The results from dietary RNAi toxicity assay demonstrate significantly impacts of Diabrotica virgifera virgifera-active dsRNAs on lady beetles under the worst-case scenario at both transcriptional and phenotypic level. Interestingly, substantial differences among the four lady beetle species were observed toward the ingested exogenous dsRNAs. CONCLUSION: Such differential response to dietary RNAi may shed light on the mechanisms underlying the mode-of-action of RNAi-based biopesticides. © 2020 Society of Chemical Industry.

Enhanced metabolism and selection of pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).

Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2-4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae).

The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the main insect pest of peppers (Capsicum spp.) throughout the southern U.S. and a potential target for novel control methods that may require gene expression analyses. Careful selection of adequate reference genes to normalize RT-qPCR data is an important prerequisite for gene expression studies since the expression stability of reference genes can be affected by the experimental conditions leading to biased or erroneous results. The lack of studies on validation of reference genes for RT-qPCR analysis in A. eugenii limits the investigation of gene expression, therefore it is needed a systematic selection of suitable reference genes for data normalization. In the present study, three programs (BestKeeper, geNorm and NormFinder) were used to analyze the expression stability of candidate reference genes (ß-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) in A. eugenii under different experimental conditions. Our results revealed that the most stably expressed reference genes in A. eugenii varied according to the experimental condition evaluated: developmental stages (EF1-α, 18S and RPL12), sex (RPS23 and RPL12), low temperature (GAPDH and α-TUB), high temperature (α-TUB and RPS23), all temperatures (α-TUB and GAPDH), starvation (RPL12 and α-TUB), and dsRNA exposure (α-TUB and RPL12). Our study provides for the first time valuable information on appropriate reference genes that can be used in the analysis of gene expression by RT-qPCR in biological experiments involving A. eugenii.

Delivery of gene-specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall.

BACKGROUND: RNA interference (RNAi) is widely used in entomological research for functional analysis of genes and is being considered as a new tool for insect pest management. Sri Lanka weevil (SLW) is a highly polyphagous pest of agronomically important plants, but currently only a few control methods are available for this insect. RESULTS: In the present study, we evaluated the stability of candidate reference genes ß-ACT, α-TUB, EF1-α, RPL12 and GAPDH, and identified EF1-α as the most reliable for gene expression normalization. Four target genes involved in different cellular processes, including Prosα2, RPS13, Snf7 and V-ATPase A were selected to evaluate whether RNAi response in SLW adults can be triggered by microinjection and oral feeding of their double-stranded RNAs (dsRNAs). Three days after injection of the dsRNAs for the target genes, their transcript levels were significantly reduced (up to 91.4%) when compared to the control. Additionally, weevils fed with the target dsRNAs showed significant decreases in gene transcript levels and significant mortality was observed in insects treated with Prosα2 and Snf7 dsRNAs (78.6 to 92.7%). CONCLUSION: Our data demonstrate that microinjection and feeding of dsRNA produce a strong RNAi response in SLW, indicating that RNAi-based strategies could be explored to develop a selective and environmentally safe control method against SLW. © 2019 Society of Chemical Industry.

Control of western corn rootworm via RNAi traits in maize: lethal and sublethal effects of Sec23 dsRNA.

BACKGROUND: RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins. RESULTS: Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays. CONCLUSION: We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry.

Transcriptional responses of soybean aphids to sublethal insecticide exposure.

Insecticides are a key tool in the management of many insect pests of agriculture, including soybean aphids. The selection imposed by insecticide use has often lead to the evolution of resistance by the target pest through enhanced detoxification mechanisms. We hypothesised that exposure of insecticide-susceptible aphids to sublethal doses of insecticides would result in the up-regulation of genes involved in detoxification of insecticides, revealing the genes upon which selection might act in the field. We used the soybean aphid biotype 1 reference genome, version 6.0 as a reference to analyze RNA-Seq data. We identified multiple genes with potential detoxification roles that were up-regulated 12 h after sublethal exposure to esfenvalerate or thiamethoxam. However, these genes were part of a dramatic burst of differential gene expression in which thousands of genes were up- or down-regulated, rather than a defined response to insecticides. Interestingly, the transcriptional burst observed at 12 h s declined dramatically by 24-hrs post-exposure, suggesting a general stress response that may become fine-tuned over time.

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber).

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), ß-actin (Actin), ß-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.

Responses of two ladybird beetle species (Coleoptera: Coccinellidae) to dietary RNAi.

BACKGROUND: One concern with the adoption of RNAi-based genetically engineered (GE) crops is the potential harm to valued non-target organisms. Species of Coccinellidae (Coleoptera) are important natural enemies and might be exposed to the insecticidal dsRNA produced by the plant. To assess their susceptibility to dietary RNAi, we fed Adalia bipunctata and Coccinella septempunctata with a dsRNA designed to target the vATPase A of the western corn rootworm, Diabrotica virgifera virgifera (Dvv dsRNA). Specific dsRNAs designed to target the vATPase A of the two ladybird beetle species served as positive controls. RESULTS: Our results revealed that both species were sensitive to dietary RNAi when ingesting their own dsRNAs, with C. septempunctata being more sensitive than A. bipunctata. Dvv dsRNA also adversely affected the two ladybird beetles as indicated by a significantly (but marginally) prolonged developmental time for A. bipunctata and a significantly reduced survival rate for C. septempunctata. These results, however, were obtained at Dvv dsRNA concentrations that were orders of magnitude higher than expected to occur in the field. Gene expression analyses confirmed the bioactivity of the dsRNA treatments and the results from the feeding bioassays. These results are consistent with the bioinformatics analyses, which revealed a higher number of 21-nucleotide-long matches, a requirement for effective RNAi, of the Dvv dsRNA with the vATPase A of C. septempunctata (34 matches) than with that of A. bipunctata (six matches). CONCLUSION: Feeding bioassays revealed that two ladybird species are responsive to dietary RNAi. The two species, however, differed in their sensitivity. © 2019 Society of Chemical Industry.

Southern Corn Rootworm (Coleoptera: Chrysomelidae) Adult Emergence and Population Growth Assessment After Selection With Vacuolar ATPase-A double-stranded RNA Over Multiple Generations.

The southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), was exposed over multiple generations to vacuolar (v)ATPase-A double-stranded (ds)RNA, first as adults and later, as neonate larvae. During adult selection, high mortality and lower fecundity were observed in the RNAi-selected cages after beetles were exposed to sublethal dsRNA concentrations that varied between LC40 and LC75. During larval selection, a delay in adult emergence and effects on population growth parameters were observed after neonates were exposed to sublethal dsRNA concentrations that varied between LC50 and LC70. Some of the parameters measured for adult emergence such as time to reach maximum linear adult emergence, time elapsed before attaining linear emergence, termination point of the linear emergence, and total days of linear emergence increase, were significantly different between RNAi-selected and control colonies for at least one generation. Significant differences were also observed in population growth parameters such as growth rate, net reproductive rate, doubling time, and generation time. After seven generations of selection, there was no indication that resistance evolved. The sublethal effects caused by exposures of southern corn rootworm to dsRNAs can affect important life history traits and fitness especially through delays in adult emergence and reduction in population growth. Although changes in susceptibility did not occur, the observation of sublethal effects suggests important responses to potential selection pressure. Assuming resistance involves a recessive trait, random mating between susceptible and resistant individuals is an important factor that allows sustainable use of transgenic plants, and delays in adult emergence observed in our studies could potentially compromise this assumption.